Discovering new therapies for metastatic melanoma is still a pressing issue, given the lack of treatment options for wild type BRAF melanoma, frequent relapses and (with some drugs) limited responders, even with the recent exciting FDA approved drugs. The long-term goal of this project is to develop alternative treatments, which will lead to longer lasting clinical responses and better patient outcomes for patients with malignant melanomas. Specifically, this proposal aims to discover treatments that target anti-apoptotic defenses and pathways used by melanoma to resist apoptosis. Cancer initiating cells (CICs) are implicated in cancer cells' resistance to treatment, resulting in relapse. It is therefore crucial to identify treatment strateies that eliminate the bulk of melanoma cells (de-bulk) as well as the CIC populations of the melanoma (also known as Melanoma Initiating Cells, MICs). The BCL-2 family is important in regulating apoptosis. The interactions among the pro- and anti-apoptotic BCL-2 family members can control the initiation of intrinsic apoptosis. ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins BCL-2/BCL-XL/BCL-W. We have identified two promising agents, the synthetic retinoid derivative, fenretinide (4-HPR) and a Gamma Secretase Inhibitor (GSI-I), that synergize with ABT-737 to inhibit multiple anti-apoptotic BCL-2 family members. When tested in vitro, in melanoma cell lines and tumor cells maintained in the patient derived xenograft (PDX) model, we found that ABT-737 combined with either drug partner not only de-bulked the melanoma cells, but also killed the CICs, for melanomas with either mutant or wild-type BRAF. Both combinations also dramatically limited tumor self-renewal. MiRNA-based therapies provide an exciting, novel therapeutic opportunity for many diseases. We show that replacement of miR-26a induced cell death in a subset of melanomas, through targeting the anti-apoptotic protein, silencer of death domain (SODD). Therefore, we propose to further demonstrate the efficacy of the two approaches of targeting anti-apoptotic defenses in melanoma and investigate the mechanisms involved: inhibiting multiple anti-apoptotic BCL-2 family members at once (Aims 1 and 2) and a miRNA-based approach to target SODD (Aim 3). Aim 1: Examine the mechanism(s) determining efficacy for the combinations of ABT-737 with 4-HPR or GSI in de-bulking melanoma and targeting MICs. We will define the mechanism of action for these drug combinations to eliminate MICs using CRISPR genomic editing technology, and further examine how the combinations induced MCl-1 degradation. {Aim 2: Investigate the efficacy and mechanisms of the combination treatments of ABT-737 with 4-HPR or GSI in vivo. We will determine the effects and examine the mechanisms involved by using a conventional xenograft models with knockout cell lines, a melanoma PDX model, a melanoma metastasis model, and a low-cell-number implantation xenograft model for assessing MIC-initiated tumor formation.} {Aim 3: Study the effects of miR-26a replacement in de-bulking melanoma and targeting MICs, and identify the candidate compounds as potential partners with miR-26a treatment. We will test the effects of miR-26a in targeting MICs as in Aim 1, and screen for potential partner compounds with an apoptosis compound library and a MAPK inhibitor library.} In summary, this proposal extends our exciting preliminary studies that have identified drug combinations which overcome melanoma resistance to apoptosis. We will further test these promising combinations with effects on de-bulking melanoma, and eliminating the MIC populations and limiting their self-renewal. We will also determine the effectiveness of an original miRNA-based therapy alone or in combination. Results are likely to identify new therapeutic approaches for treating melanoma, with unique effectiveness in killing MICs.